Applied biosystems 3730 dna analyser

PCR products were purified using the QIAquick Gel Extraction Kit. Briefly, DNA fragments were excised from the agarose gel, solubilised with Buffer QG (50 °C, 10 min) before isopropanol precipitation. DNA was collected on a QIAquick spin column before eluting with dH₂0. PCR products were sequenced by the Tayside Centre for Genomic Analysis, Ninewells Hospital. PCR products (5 µl) were purified using a modification of the ExoSAP method by incubating with exonuclease I (1 U) and shrimp alkaline phosphatase (1 U) for 20 min at 37 °C before inactivation at 80 °C for 15 min. Samples were sequenced bidirectionally using the ABI BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems) and analysed on the Applied Biosystems 3730 DNA Analyser (Applied Biosystems). Sequence analysis was performed using MacVector version 12.03 (MacVector Inc., Waterbeach, Cambridge, UK).

Based on the Bovine Reference Sequence (BRS) GenBank acc. no. V00654^{26 (link)}, the primer pair MTF: 5'-GACTCAAGGAAGAAACTGC-3' and MTR: 5'-GACTCATCTAGGCATTTTCA-3'²⁷ was used to amplify a 1029 bp long segment of the mitochondrial DNA control region, in the nucleotide position range 15,792 – 69 (V00654). Amplification conditions were as follows: 100 ng genomic DNA, 1.5 mM MgCl2, 0·2 mM dNTPs, 1X reaction buffer, 0·2 μM of each primer, and 1-unit Taq DNA polymerase (Platinum, Invitrogen, Life Technologies) in a 25 μl final volume. Thermal protocol was set for an initial denaturation at 94 °C for 2.30 min, and then 35 cycles of 94 °C for 20 s, 56 °C for 30 s, 72 °C for 1.20 min, followed by 72 °C for 5 min were carried out. Amplicons were purified with the ChargeSwitch PCR Clean-Up Kit (Invitrogen, Carlsbad, CA, USA) and then used to perform Sanger sequencing reactions with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were run in an Applied Biosystems 3730 DNA Analyser (Applied Biosystems, Foster City, CA, USA). Sequencing reactions yielded lower then expected length in many samples, then to make sure that the same fragment is analysed in all individuals, sequences were trimmed to 616 bp. All sequences were submitted to GenBank and given accession numbers KX923119 to KX923319.